Target analyte sensors utilizing microspheres

ABSTRACT

A microsphere-based analytic chemistry system and method for making the same is disclosed in which microspheres or particles carrying bioactive agents may be combined randomly or in ordered fashion and dispersed on a substrate to form an array while maintaining the ability to identify the location of bioactive agents and particles within the array using an optically interrogatable, optical signature encoding scheme. A wide variety of modified substrates may be employed which provide either discrete or non-discrete sites for accommodating the microspheres in either random or patterned distributions. The substrates may be constructed from a variety of materials to form either two-dimensional or three-dimensional configurations. In a preferred embodiment, a modified fiber optic bundle or array is employed as a substrate to produce a high density array. The disclosed system and method have utility for detecting target analytes and screening large libraries of bioactive agents.

This application is a continuation of Application No. 09/151,877 filedSep. 11, 1998, now U.S. Pat. No. 6,327,410 which is acontinuation-in-part of application U.S. Ser. No. 08/818,199, filed Mar.14, 1997 now U.S. Pat. No. 6,023,540.

This invention was made with government support under N00014-95-1-1340awarded by the Department of the Navy, Office of Naval Research. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The use of optical fibers and optical fiber strands in combination withlight absorbing dyes for chemical analytical determinations hasundergone rapid development, particularly within the last decade. Theuse of optical fibers for such purposes and techniques is described byMilanovich et al., “Novel Optical Fiber Techniques For MedicalApplication”, Proceedings of the SPIE 28th Annual InternationalTechnical Symposium On Optics and Electro-Optics, Volume 494, 1980;Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators andFiber Optics” in C.R.C. Critical Reviews In Analytical Chemistry, Vol.19, 1988, pp. 135-173; Wolfbeis, O. S., “Fiber Optical Fluorosensors InAnalytical Chemistry” in Molecular Luminescence Spectroscopy, Methodsand Applications (S. G. Schulman, editor), Wiley & Sons, New York(1988); Angel, S. M., Spectroscopy 2 (4):38 (1987); Walt, et al.,“Chemical Sensors and Microinstrumentation”, ACS Symposium Series, Vol.403, 1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors,Ed. CRC Press, Boca Raton, Fla., 1991, 2nd Volume.

When using an optical fiber in an in vitro/in vivo sensor, one or morelight absorbing dyes are located near its distal end. Typically, lightfrom an appropriate source is used to illuminate the dyes through thefiber's proximal end. The light propagates along the length of theoptical fiber; and a portion of this propagated light exits the distalend and is absorbed by the dyes. The light absorbing dye may or may notbe immobilized; may or may not be directly attached to the optical fiberitself; may or may not be suspended in a fluid sample containing one ormore analytes of interest; and may or may not be retainable forsubsequent use in a second optical determination.

Once the light has been absorbed by the dye, some light of varyingwavelength and intensity returns, conveyed through either the same fiberor collection fiber(s) to a detection system where it is observed andmeasured. The interactions between the light conveyed by the opticalfiber and the properties of the light absorbing dye provide an opticalbasis for both qualitative and quantitative determinations.

Of the many different classes of light absorbing dyes whichconventionally are employed with bundles of fiber strands and opticalfibers for different analytical purposes are those more commoncompositions that emit light after absorption termed “fluorophores” andthose which absorb light and internally convert the absorbed light toheat, rather than emit it as light, termed “chromophores.”

Fluorescence is a physical phenomenon based upon the ability of somemolecules to absorb light (photons) at specified wavelengths and thenemit light of a longer wavelength and at a lower energy. Substances ableto fluoresce share a number of common characteristics: the ability toabsorb light energy at one wavelength λ_(ab); reach an excited energystate; and subsequently emit light at another light wavelength, λ_(em).The absorption and fluorescence emission spectra are individual for eachfluorophore and are often graphically represented as two separate curvesthat are slightly overlapping. The same fluorescence emission spectrumis generally observed irrespective of the wavelength of the excitinglight and, accordingly, the wavelength and energy of the exciting lightmay be varied within limits; but the light emitted by the fluorophorewill always provide the same emission spectrum. Finally, the strength ofthe fluorescence signal may be measured as the quantum yield of lightemitted. The fluorescence quantum yield is the ratio of the number ofphotons emitted in comparison to the number of photons initiallyabsorbed by the fluorophore. For more detailed information regardingeach of these characteristics, the following references are recommended:Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press,New York, 1983; Freifelder, D., Physical Biochemistry, second edition,W. H. Freeman and Company, New York, 1982; “Molecular LuminescenceSpectroscopy Methods and Applications: Part I” (S. G. Schulman, editor)in Chemical Analysis, vol. 77, Wiley & Sons, Inc., 1985; The Theory ofLuminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London,1968.

In comparison, substances which absorb light and do not fluoresceusually convert the light into heat or kinetic energy. The ability tointernally convert the absorbed light identifies the dye as a“chromophore.” Dyes which absorb light energy as chromophores do so atindividual wavelengths of energy and are characterized by a distinctivemolar absorption coefficient at that wavelength. Chemical analysisemploying fiber optic strands, bundles, or arrays and absorptionspectroscopy using visible and ultraviolet light wavelengths incombination with the absorption coefficient allow for the determinationof concentration for specific analyses of interest by spectralmeasurement. The most common use of absorbance measurement via opticalfibers is to determine concentration which is calculated in accordancewith Beers' law; accordingly, at a single absorbance wavelength, thegreater the quantity of the composition which absorbs light energy at agiven wavelength, the greater the optical density for the sample. Inthis way, the total quantity of light absorbed directly correlates withthe quantity of the composition in the sample.

Many of the recent improvements employing optical fiber sensors in bothqualitative and quantitative analytical determinations concern thedesirability of depositing and/or immobilizing various light absorbingdyes at the distal end of the optical fiber. In this manner, a varietyof different optical fiber chemical sensors and methods have beenreported for specific analytical determinations and applications such aspH measurement, oxygen detection, and carbon dioxide analyses. Thesedevelopments are exemplified by the following publications: Freeman, etal., Anal Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205:1,(1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan, et al.,Anal. Chem. 59:437 (1987); Lubbers et al., Sens. Actuators 1983;Munkholm et al., Talanta 35:109 (1988); Munkholm et al., Anal. Chem.58:1427 (1986); Seitz, W. R., Anal. Chem. 56:16A-34A (1984); Peterson,et al., Anal. Chem. 52:864 (1980): Saari, et al., Anal. Chem. 54:821(1982); Saari, et al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal.Chem. Acta. 160:47 (1984); Schwab, et al., Anal. Chem. 56:2199 (1984);Wolfbeis, O. S., “Fiber Optic Chemical Sensors”, Ed. CRC Press, BocaRaton, Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R., Anal.Chem., 481A-487A, Vol. 67, (1995).

More recently, fiber optic sensors have been constructed that permit theuse of multiple dyes with a single, discrete fiber optic strand, fiberoptic bundles or fiber optic arrays, such as imaging fibers. U.S. Pat.Nos. 5,244,636 and 5,250,264 to Walt, et al. disclose systems foraffixing multiple, different dyes on the distal end of the bundle, theteachings of each of these patents being incorporated herein by thisreference. The disclosed configurations enable separate optical fibersof the bundle to optically access individual dyes. This avoids theproblem of deconvolving the separate signals in the returning light fromeach dye, which arises when the signals from two or more dyes arecombined, each dye being sensitive to a different analyte, and there issignificant overlap in the dyes' emission spectra.

The innovation of the two previous patents was the placement of multiplechemical functionalities at the end of a single optical fiber bundlesensor. This configuration yielded an analytic chemistry sensor thatcould be remotely monitored via the typically small bundle. Thedrawback, however, was the difficulty in applying the variouschemistries associated with the chemical functionalities at the sensor'send; the functionalities were built on the sensor's end in a serialfashion. This was a slow process, and in practice, only tens offunctionalities could be applied. Accordingly, compositions and methodsare desirable that allow the generation of large fiber optic arraysincluding microspheres that can be either encoded or decoded to allowthe detection of target analytes.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention providescompositions comprising a substrate with a surface comprising discretesites, and a population of microspheres distributed on the sites. Thesites may be wells or chemically functionalized sites.

In an additional aspect the invention provides methods of determiningthe presence of a target analyte in a sample. The methods comprisecontacting the sample with a composition comprising a substrate with asurface comprising discrete sites, and a population of microspherescomprising at least a first and a second subpopulation. Eachsubpopulation comprises a bioactive agent and an optical signaturecapable of identifying the bioactive agent. The microspheres aredistributed on the surface such that the discrete sites containmicrospheres. The presence or absence of the target analyte is thendetermined.

In a further aspect, the invention provides methods of making acomposition comprising forming a surface comprising individual sites ona substrate, distributing microspheres on the surface such that theindividual sites contain microspheres. The microspheres comprise atleast a first and a second subpopulation, each comprising a bioactiveagent, and an optical signature capable of identifying said bioactiveagent.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, reference characters refer to the sameparts throughout the different views. The drawings are not necessarilyto scale; emphasis has instead been placed upon illustrating theprinciples of the invention. Of the drawings:

FIG. 1 is a schematic diagram illustrating the optical signatureencoding and chemical functionalizing of the microspheres according tothe present invention;

FIG. 2 is a process diagram describing the preparation, encoding, andfunctionalizing of the microspheres of the present invention;

FIG. 3 is a schematic diagram illustrating a microsphere systemincluding microspheres with different chemical functionalities andencoded descriptions of the functionalities;

FIG. 4 is a schematic diagram of the inventive fiber optic sensor andassociated instrumentation and control system;

FIGS. 5A and 5B are micrographs illustrating the preferred technique forattaching or affixing the microspheres to the distal end of the opticalfiber bundle;

FIG. 6 is a process diagram describing well formation in the opticalfiber bundle and affixation of the microspheres in the wells;

FIGS. 7A and 7B are micrographs showing the array of microspheres intheir corresponding wells prior and subsequent to physical agitation,tapping and air pulsing, demonstrating the electrostatic binding of themicrospheres in the wells;

FIGS. 8A, 8B, and 8C are micrographs from alkaline phosphatasemicrospheres when exposed to fluorescein diphosphate, at the fluoresceinemission wavelength, at an encoding wavelength for DiIC, and at anencoding wavelength for TRC, respectively;

FIGS. 9A and 9B are micrographs showing the optical signal fromβ-galactosidase microspheres when exposed to fluoresceinβ-galactopyranoside at the fluorescein emission wavelength and at anencoding wavelength for DiIC, respectively; and

FIGS. 10A and 10B are micrographs showing the optical response fromrabbit antibody microspheres prior to and post, respectively, exposureto fluorescein labeled antigens.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on two synergistic inventions: 1) thedevelopment of a bead-based analytic chemistry system in which beads,also termed microspheres, carrying different chemical functionalitiesmay be mixed together while the ability is retained to identify thefunctionality of each bead using an optically interrogatable encodingscheme (an “optical signature”); and 2) the use of a substratecomprising a patterned surface containing individual sites that can bindor associate individual beads. This allows the synthesis of thebioactive agents (i.e. compounds such as nucleic acids and antibodies)to be separated from their placement on an array, i.e. the bioactiveagents may be synthesized on the beads, and then the beads are randomlydistributed on a patterned surface. Since the beads are first coded withan optical signature, this means that the array can later be “decoded”,i.e. after the array is made, a correlation of the location of anindividual site on the array with the bead or bioactive agent at thatparticular site can be made. This means that the beads may be randomlydistributed on the array, a fast and inexpensive process as compared toeither the in situ synthesis or spotting techniques of the prior art.Once the array is loaded with the beads, the array can be decoded, orcan be used, with full or partial decoding occurring after testing, asis more fully outlined below.

Accordingly, the present invention provides array compositionscomprising at least a first substrate with a surface comprisingindividual sites. By “array” herein is meant a plurality of bioactiveagents in an array format; the size of the array will depend on thecomposition and end use of the array. Arrays containing from about 2different bioactive agents (i.e. different beads) to many millions canbe made, with very large fiber optic arrays being possible. Generally,the array will comprise from two to as many as a billion or more,depending on the size of the beads and the substrate, as well as the enduse of the array, thus very high density, high density, moderatedensity, low density and very low density arrays may be made. Preferredranges for very high density arrays are from about 10,000,000 to about2,000,000,000, with from about 100,000,000 to about 1,000,000,000 beingpreferred. High density arrays range about 100,000 to about 10,000,000,with from about 1,000,000 to about 5,000,000 being particularlypreferred. Moderate density arrays range from about 10,000 to about50,000 being particularly preferred, and from about 20,000 to about30,000 being especially preferred. Low density arrays are generally lessthan 10,000, with from about 1,000 to about 5,000 being preferred. Verylow density arrays are less than 1,000, with from about 10 to about 1000being preferred, and from about 100 to about 500 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single bioactive agent may be made as well. In addition, insome arrays, multiple substrates may be used, either of different oridentical compositions. Thus for example, large arrays may comprise aplurality of smaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 nmcan be used, and very small fibers are known, it is possible to have asmany as 250,000 different fibers and beads in a 1 mm² fiber optic arrayor bundle, with densities of greater than 15,000,000 individual beadsand fibers per 0.5 cm² obtainable.

The compositions comprise a substrate. By “substrate” or “solid support”or other grammatical equivalents herein is meant any material that canbe modified to contain discrete individual sites appropriate for theattachment or association of beads and is amenable to at least onedetection method. As will be appreciated by those in the art, the numberof possible substrates are very large, and include, but are not limitedto, glass and modified or functionalized glass, plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,etc.), polysaccharides, nylon or nitrocellulose, resins, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses, plastics, optical fiber bundles, and avariety of other polymers. In general, the substrates allow opticaldetection and do not appreciably fluoresce.

Generally the substrate is planar, although as will be appreciated bythose in the art, other configurations of substrates may be used aswell; for example, three dimensional configurations can be used, forexample by embedding the beads in a porous block of plastic that allowssample access to the beads and using a confocal microscope fordetection. Similarly, the beads may be placed on the inside surface of atube, for flow-through sample analysis to minimize sample volume.Preferred substrates include optical fiber bundles as discussed below,and flat planar substrates such as glass, polystyrene and other plasticsand acrylics.

At least one surface of the substrate is modified to contain discrete,individual sites for later association of microspheres. These sites maycomprise physically altered sites, i.e. physical configurations such aswells or small depressions in the substrate that can retain the beads,such that a microsphere can rest in the well, or the use of other forces(magnetic or compressive), or chemically altered or active sites, suchas chemically functionalized sites, electrostatically altered sites,hydrophobically/hydrophilically functionalized sites, spots of adhesive,etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the substrate.However, it should be noted that these sites may not be discrete sites.That is, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the attachment of beads at anyposition. That is, the surface of the substrate is modified to allowattachment of the microspheres at individual sites, whether or not thosesites are contiguous or non-contiguous with other sites. Thus, thesurface of the substrate may be modified such that discrete sites areformed that can only have a single associated bead, or alternatively,the surface of the substrate is modified and beads may go down anywhere,but they end up at discrete sites.

In a preferred embodiment, the surface of the substrate is modified tocontain wells, i.e. depressions in the surface of the substrate. Thismay be done as is generally known in the art using a variety oftechniques, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. As will beappreciated by those in the art, the technique used will depend on thecomposition and shape of the substrate.

In a preferred embodiment, physical alterations are made in a surface ofthe substrate to produce the sites. In a preferred embodiment, thesubstrate is a fiber optic bundle or array and the surface of thesubstrate is a terminal end of the fiber bundle or array. In thisembodiment, wells are made in a terminal or distal end of a fiber opticbundle or array comprising individual fibers. In this embodiment, thecores of the individual fibers are etched, with respect to the cladding,such that small wells or depressions are formed at one end of thefibers. The required depth of the wells will depend on the size of thebeads to be added to the wells.

Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a preferred embodiment, the surface of the substrate is modified tocontain chemically modified sites, that can be used to attach, eithercovalently or non-covalently, the microspheres of the invention to thediscrete sites or locations on the substrate. “Chemically modifiedsites” in this context includes, but is not limited to, the addition ofa pattern of chemical functional groups including amino groups, carboxygroups, oxo groups and thiol groups, that can be used to covalentlyattach microspheres, which generally also contain corresponding reactivefunctional groups; the addition of a pattern of adhesive that can beused to bind the microspheres (either by prior chemicalfunctionalization for the addition of the adhesive or direct addition ofthe adhesive); the addition of a pattern of charged groups (similar tothe chemical functionalities) for the electrostatic attachment of themicrospheres, i.e. when the microspheres comprise charged groupsopposite to the sites; the addition of a pattern of chemical functionalgroups that renders the sites differentially hydrophobic or hydrophilic,such that the addition of similarly hydrophobic or hydrophilicmicrospheres under suitable experimental conditions will result inassociation of the microspheres to the sites on the basis ofhydroaffinity. For example, the use of hydrophobic sites withhydrophobic beads, in an aqueous system, drives the association of thebeads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

The compositions of the invention further comprise a population ofmicrospheres. By “population” herein is meant a plurality of beads asoutlined above for arrays. Within the population are separatesubpopulations, which can be a single microsphere or multiple identicalmicrospheres. That is, in some embodiments, as is more fully outlinedbelow, the array may contain only a single bead for each bioactiveagent; preferred embodiments utilize a plurality of beads of each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of bioactive agent and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphited, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either bioactive agent attachment or tagattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

FIG. 1 illustrates the construction of a bead or microsphere 10according to the principles of the present invention. In common with theprior art, the microsphere 10 is given a bioactive agent 12, which istypically applied to the microsphere's surface. The bioactive agent isdesigned so that in the presence of the analyte(s) to which it istargeted, an optical signature of the microsphere, possibly includingregion surrounding it, is changed.

It should be noted that a key component of the invention is the use of asubstrate/bead pairing that allows the association or attachment of thebeads at discrete sites on the surface of the substrate, such that thebeads do not move during the course of the assay.

Each microsphere comprises two components: a bioactive agent and anoptical signature. By “candidate bioactive agent” or “bioactive agent”or “chemical functionality” or “binding ligand” herein is meant as usedherein describes any molecule, e.g., protein, oligopeptide, smallorganic molecule, polysaccharide, polynucleotide, etc. which can beattached to the microspheres of the invention. It should be understoodthat the compositions of the invention have two primary uses. In apreferred embodiment, as is more fully outlined below, the compositionsare used to detect the presence of a particular target analyte; forexample, the presence or absence of a particular nucleotide sequence ora particular protein, such as an enzyme, an antibody or an antigen. Inan alternate preferred embodiment, the compositions are used to screenbioactive agents, i.e. drug candidates, for binding to a particulartarget analyte.

Bioactive agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Bioactive agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The bioactiveagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Bioactive agents are also found amongbiomolecules including peptides, nucleic acids, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof. Particularly preferred are nucleic acids andproteins.

Bioactive agents can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification and/oramidification to produce structural analogs.

In a preferred embodiment, the bioactive agents are proteins. By“protein” herein is meant at least two covalently attached amino acids,which includes proteins, polypeptides, oligopeptides and peptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein means both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnorleucine are considered amino acids for the purposes of the invention.The side chains may be in either the (R) or the (S) configuration. Inthe preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations.

In one preferred embodiment, the bioactive agents are naturallyoccurring proteins or fragments of naturally occuring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the bioactive agents are peptides of fromabout 5 to about 30 amino acids, with from about 5 to about 20 aminoacids being preferred, and from about 7 to about 15 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized bioactive proteinaceous agents.

In a preferred embodiment, a library of bioactive agents are used. Thelibrary should provide a sufficiently structurally diverse population ofbioactive agents to effect a probabilistically sufficient range ofbinding to target analytes. Accordingly, an interaction library must belarge enough so that at least one of its members will have a structurethat gives it affinity for the target analyte. Although it is difficultto gauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. Thus, in a preferred embodiment, at least 10⁶, preferably atleast 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹different bioactive agents are simultaneously analyzed in the subjectmethods. Preferred methods maximize library size and diversity.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bioactive agents are nucleic acids(generally called “probe nucleic acids” or “candidate probes” herein).By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl,et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. AcidsRes., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al., ChemicaScripta, 26:141 (1986)), phosphorothioate (Mag, et al., Nucleic AcidsRes., 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu, et al., J. Am. Chem. Soc., 111:2321 (1989)),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.,114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207(1996), all of which are incorporated by reference)). Other analognucleic acids include those with positive backbones (Denpcy, et al.,Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S.Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863;Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991);Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); Letsinger, etal., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xanthanine, hypoxanthanine, isocytosine, isoguanine, and basepairanalogs such as nitropyrrole and nitroindole, etc.

As described above generally for proteins, nucleic acid bioactive agentsmay be naturally occuring nucleic acids, random nucleic acids, or“biased” random nucleic acids. For example, digests of procaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In general, probes of the present invention are designed to becomplementary to a target sequence (either the target analyte sequenceof the sample or to other probe sequences, as is described herein), suchthat hybridization of the target and the probes of the present inventionoccurs. This complementarity need not be perfect; there may be anynumber of base pair mismatches that will interfere with hybridizationbetween the target sequence and the single stranded nucleic acids of thepresent invention. However, if the number of mutations is so great thatno hybridization can occur under even the least stringent ofhybridization conditions, the sequence is not a complementary targetsequence. Thus, by “substantially complementary” herein is meant thatthe probes are sufficiently complementary to the target sequences tohybridize under the selected reaction conditions. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. In another embodiment, less stringenthybridization conditions are used; for example, moderate or lowstringency conditions may be used, as are known in the art; see Maniatisand Ausubel, supra, and Tijssen, supra.

The term “target sequence” or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA and rRNA, or others. It may be any length, withthe understanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart.

In a preferred embodiment, the bioactive agents are organic chemicalmoieties, a wide variety of which are available in the literature.

In a preferred embodiment, each bead comprises a single type ofbioactive agent, although a plurality of individual bioactive agents arepreferably attached to each bead. Similarly, preferred embodimentsutilize more than one microsphere containing a unique bioactive agent;that is, there is redundancy built into the system by the use ofsubpopulations of microspheres, each microsphere in the subpopulationcontaining the same bioactive agent.

As will be appreciated by those in the art, the bioactive agents mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the bioactive agents to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the bioactive agents are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, such as peptides, organicmoieties, and nucleic acids. It is a relatively straightforward matterto adjust the current synthetic techniques to use beads.

In a preferred embodiment, the bioactive agents are synthesized first,and then covalently attached to the beads. As will be appreciated bythose in the art, this will be done depending on the composition of thebioactive agents and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres are listed in Table I.

TABLE I Surface chemistry Name: NH₂ Amine COOH Carboxylic Acid CHOAldehyde CH₂—NH₂ Aliphalic Amine CO NH₂ Amide CH₂—Cl ChloromethylCONH—NH₂ Hydrazide OH Hydroxyl SO₄ Sulfate SO₃ Sulfonate Ar NH₂ AromaticAmine

These functional groups can be used to add any number of differentbioactive agents to the beads, generally using known chemistries. Forexample, bioactive agents containing carbohydrates may be attached to anamino-functionalized support; the aldehyde of the carbohydrate is madeusing standard techniques, and then the aldehyde is reacted with anamino group on the surface. In an alternative embodiment, a sulfhydryllinker may be used. There are a number of sulfhydryl reactive linkersknown in the art such as SPDP, maleimides, α-haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference) which can be used to attach cysteine containingproteinaceous agents to the support. Alternatively, an amino group onthe bioactive agent may be used for attachment to an amino group on thesurface. For example, a large number of stable bifunctional groups arewell known in the art, including homobifunctional and heterobifunctionallinkers (see Pierce Catalog and Handbook, pages 155-200). In anadditional embodiment, carboxyl groups (either from the surface or fromthe bioactive agent) may be derivatized using well known linkers (seethe Pierce catalog). For example, carbodiimides activate carboxyl groupsfor attack by good nucleophiles such as amines (see Torchilin et al.,Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991),expressly incorporated herein). Proteinaceous bioactive agents may alsobe attached using other techniques known in the art, for example for theattachment of antibodies to polymers; see Slinkin et al., Bioconj. Chem.2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994);and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994), all of which arehereby expressly incorporated by reference). It should be understoodthat the bioactive agents may be attached in a variety of ways,including those listed above. What is important is that manner ofattachment does not significantly alter the functionality of thebioactive agent; that is, the bioactive agent should be attached in sucha flexible manner as to allow its interaction with a target.

Specific techniques for immobilizing enzymes on microspheres are knownin the prior art. In one case, NH₂ surface chemistry microspheres areused. Surface activation is achieved with a 2.5% glutaraldehyde inphosphate buffered saline (10 mM) providing a pH of 6.9. (138 mM NaCl,2.7 mM, KCl). This is stirred on a stir bed for approximately 2 hours atroom temperature. The microspheres are then rinsed with ultrapure waterplus 0.01% tween 20 (surfactant) −0.02%, and rinsed again with a pH 7.7PBS plus 0.01% tween 20. Finally, the enzyme is added to the solution,preferably after being prefiltered using a 0.45 μm amicon micropurefilter.

In addition to a bioactive agent, the microspheres comprise an opticalsignature that can be used to identify the attached bioactive agent.That is, each subpopulation of microspheres comprise a unique opticalsignature or optical tag that can be used to identify the uniquebioactive agent of that subpopulation of microspheres; a bead comprisingthe unique optical signature may be distinguished from beads at otherlocations with different optical signatures. As is outlined herein, eachbioactive agent will have an associated unique optical signature suchthat any microspheres comprising that bioactive agent will beidentifiable on the basis of the signature. As is more fully outlinedbelow, it is possible to reuse or duplicate optical signatures within anarray, for example, when another level of identification is used, forexample when beads of different sizes are used, or when the array isloaded sequentially with different batches of beads.

In a preferred embodiment, the optical signature is generally a mixtureof reporter dyes, preferably fluoroscent. By varying both thecomposition of the mixture (i.e. the ratio of one dye to another) andthe concentration of the dye (leading to differences in signalintensity), matrices of unique tags may be generated. This may be doneby covalently attaching the dyes to the surface of the beads, oralternatively, by entrapping the dye within the bead. The dyes may bechromophores or phosphors but are preferably fluorescent dyes, which dueto their strong signals provide a good signal-to-noise ratio fordecoding. Suitable dyes for use in the invention include, but are notlimited to, fluorescent lanthanide complexes, including those ofEuropium and Terbium, fluorescein, rhodamine, tetramethylrhodamine,eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, and others describedin the 1989-1991 Molecular Probes Handbook by Richard P. Haugland,hereby expressly incorporated by reference.

In a preferred embodiment, the encoding can be accomplished in a ratioof at least two dyes, although more encoding dimensions may be added inthe size of the beads, for example. In addition, the labels aredistinguishable from one another; thus two different labels may comprisedifferent molecules (i.e. two different fluors) or, alternatively, onelabel at two different concentrations or intensity.

In a preferred embodiment, the dyes are covalently attached to thesurface of the beads. This may be done as is generally outlined for theattachment of the bioactive agents, using functional groups on thesurface of the beads. As will be appreciated by those in the art, theseattachments are done to minimize the effect on the dye.

In a preferred embodiment, the dyes are non-covalently associated withthe beads, generally by entrapping the dyes in the bead matrix or poresof the beads. Referring to the embodiment of FIG. 1, reporter dyes 14are added to the microsphere 10 with the encoding occurring in the ratioof two or more dyes. The reporter dyes 14 may be chromophore-type.Fluorescent dyes, however, are preferred because the strength of thefluorescent signal provides a better signal-to-noise ratio whendecoding. Additionally, encoding in the ratios of the two or more dyes,rather than single dye concentrations, is preferred since it providesinsensitivity to the intensity of light used to interrogate the reporterdye's signature and detector sensitivity. In one embodiment, the dyesare added to the bioactive agent, rather than the beads, although thisis generally not preferred.

FIG. 2 is a process diagram illustrating the preparation of themicrospheres. In step 50, an aliquot of stock microspheres are vacuumfiltered to produce a dry cake. In one implementation, microspherecopolymers of methylstyrene (87%) and divinylbenzene (13%) are used thathave a 3.1 micrometer (μm) diameter. The dry cake is then broken apartand a dye solution added to it in step 52 to encode optical signaturesof the microspheres with information concerning the intended surfacechemical functionalities. Dyes may be covalently bonded to themicrospheres' surface, but this consumes surface binding sites desirablyreserved for the chemical functionalities. Preferably, the microspheresare placed in a dye solution comprising a ratio of two or morefluorescent reporter dyes dissolved in an organic solvent that willswell the microspheres, e.g., dimethylformamide (DMF). The length oftime the microspheres are soaked in the dye solution will determinetheir intensity and the broadness of the ratio range.

In an exemplary two dye system, Texas Red Cadaverine (TRC) is used,which is excited at λ_(ab)=580 mm and emits at λ_(em)=630 mm, incombination with indodicarbocyanine (DiIC): 610/670 (λ_(ab)/λ_(em)).Generally, dyes are selected to be compatible with the chemistriesinvolved in the analysis and to be spectrally compatible. This avoidsdeconvolution problems associated with determining signal contributionsbased on the presence of both the analyte and the encoding dye ratioscontributing to an overlapping emission spectral region.

Examples of other dyes that can be used are Oxazin (662/705), IR-144(745/825), IR-140 (776/882), IR-125 (786/800) from Exciton, and Bodipy665/676 from Molecular Probes, and Naphthofluorescein (605/675) alsofrom Molecular Probes. Lathanide complexes may also be used. Fluorescentdyes emitting in other than the near infrared may also be used.Chromophore dyes are still another alternative that produce an opticallyinterrogatable signature, as are more exotic formulations using Ramanscattering-based dyes or polarizing dyes, for example. The ability of aparticular dye pair to encode for different chemical functionalitiesdepends on the resolution of the ratiometric measurement.Conservatively, any dye pair should provide the ability to discriminateat least twenty different ratios. The number of unique combinations oftwo dyes made with a particular dye set is shown in the following TableII.

TABLE II NUMBER OF COMBINATIONS DYES IN SET POSSIBLE 3 3 4 6 5 10 6 15

Thus, using six dyes and twenty distinct ratios for each dye pair, 300separate chemical functionalities may be encoded in a given populationof microspheres. Combining more than two dyes provides additionaldiversity in the encoding combinations. Furthermore, the concentrationof the dyes will contribute to their intensity; thus intensity isanother way to increase the number of unique optical signatures.

In step 54, the microspheres are vacuum filtered to remove excess dye.The microspheres are then washed in water or other liquid that does notswell the microspheres, but in which the dyes are still soluble. Thisallows the residual dye to be rinsed off without rinsing the dye out ofthe microspheres.

In step 56, the bioactive agent is attached to the microsphere surfaceif not already present. It should be understood that surface chemistriesmay be present throughout the microsphere's volume, and not limited tothe physical circumferential surface. Once the microspheres are madecomprising at least one bioactive agent and an optical signature, themicrospheres are added to discrete sites on the surface of thesubstrate. This can be done in a number of ways, but generally comprisesadding the beads to the surface under conditions that will allow theassociation of the microspheres on or at the discrete sites. Theassociation of the beads on the surface may comprise a covalent bondingof the bead to the surface, for example when chemical attachment sitesare added to both the substrate and the bead; an electrostatic orhydroaffinity, when charge, hydrophobicity or hydrophilicity is used asthe basis of the binding; a physical yet non-covalent attachment such asthe use of an adhesive; or a spatial attachment, for example thelocalization of a bead within a well. In some embodiments it may bepreferable to effect a more permanent attachment after the initiallocalization, for example through the use of cross-linking agents, afilm or membrane over the array.

FIG. 3 schematically illustrates a microsphere system, or array ofmicrospheres, 100 formed from microsphere populations that havedifferent bioactive agents. While a large number of microspheres andbioactive agents may be employed, in this example only three microspherepopulations are shown. The individual populations, or subpopulations, ofmicrospheres are represented as 10 a,10 b,10 c carrying respectivebioactive agents or probe sequences 60 a,60 b,60 c, as exemplaryfunctionalities. The subpopulations may be combined in either a randomor ordered fashion on a substrate, with a corresponding distribution oftheir respective bioactive agents.

Typically, with conventional methods, mixing microsphere populationshaving different bioactive agents results in the loss of informationregarding the selectivity for each of the corresponding targetsequences. In a solution of microspheres with each of the probesequences 60 a, 60 b, and 60 c, it is possible to determine that atleast one of the target sequences 62 a, 62 b, and 62 c is present when afluorescent marker dye 64 concentration is observed on the microspheres10. However, with conventional approaches, typically there is no way todetermine which bioactive agent or probe sequence 60 a, 60 b, and 60 cis generating the activity since the information concerning whichmicrosphere contained which probe sequence was lost when thesubpopulations were mixed.

However, with the microsphere system 100 and method of the presentinvention, each microsphere in each subpopulation is encoded with acommon optical signature. In the illustrated example, the subpopulationrepresented by microsphere 10 a has a two reporter dye ratio of 10:1;the subpopulation of microspheres 10 b has a ratio of 1:1 of the samereporter dyes, and subpopulation of microspheres 10 c has a ratio of1:10 of the reporter dyes.

Thus, the randomly mixed subpopulations of microspheres are useful as ananalytic chemistry system based on each of the carried bioactive agents60 a-60 c separately. The microsphere array or system 100 is exposed toan analyte of interest to which some of the bioactive agents mayinteract. Any interaction changes the optical response of thecorresponding microspheres by, for example, binding a fluorescent dye 64to the microspheres. By identifying the chemical functionalities of themicrosphere in which the optical signature has changed, using theencoded dye combinations, information regarding the chemical identityand concentration of an analyte may be gained based upon the interactionor noninteraction of each bioactive agent contained in the microspheresystem 100.

The microspheres exhibiting activity or changes in their opticalsignature may be identified by a conventional optical train and opticaldetection system. Decoding can also be performed either manually orautomatically with the aid of a computer. Depending on the particularencoding or reporter dyes used and their operative wavelengths, opticalfilters designed for a particular wavelengths may be employed foroptical interrogation of the microspheres of bioactive agents. In apreferred embodiment, the analytic chemistry microsphere system is usedin conjunction with an optical fiber bundle or fiber optic array as asubstrate.

FIG. 4 is a schematic block diagram showing a microsphere-based analyticchemistry system employing a fiber optic assembly 200 with an opticaldetection system. The fiber optic assembly 200 comprises a fiber opticbundle or array 202, that is constructed from clad fibers so that lightdoes not mix between fibers. A microsphere array or system, 100 isattached to the bundle's distal end 212, with the proximal end 214 beingreceived by a z-translation stage 216 and x-y micropositioner 218. Thesetwo components act in concert to properly position the proximal end 214of the bundle 202 for a microscope objective lens 220. Light collectedby the objective lens 220 is passed to a reflected light fluorescenceattachment with three pointer cube slider 222. The attachment 222 allowsinsertion of light from a 75 Watt Xe lamp 224 through the objective lens220 to be coupled into the fiber bundle 202. The light from the source224 is condensed by condensing lens 226, then filtered and/or shutteredby filter and shutter wheel 228, and subsequently passes through a NDfilter slide 230.

Light returning from the distal end 212 of the bundle 202 is passed bythe attachment 222 to a magnification changer 232 which enablesadjustment of the image size of the fiber's proximal or distal end.Light passing through the magnification changer 232 is then shutteredand filtered by a second wheel 234. The light is then imaged on a chargecoupled device (CCD) camera 236. A computer 238 executes imagingprocessing software to process the information from the CCD camera 236and also possibly control the first and second shutter and filter wheels228, 234. The instrumentation exclusive of the fiber sensor 200, i.e.,to the left of the proximal end of the bundle 202 is discussed morecompletely by Bronk, et al., Anal. Chem. 1995, Vol. 67, number 17, pp.2750-2752.

The microsphere array or system 100 may be attached to the distal end ofthe optical fiber bundle or fiber optic array using a variety ofcompatible processes. It is important that the microspheres are locatedclose to the end of the bundle. This ensures that the light returning ineach optical fiber predominantly comes from only a single microsphere.This feature is necessary to enable the interrogation of the opticalsignature of individual microspheres to identify reactions involving themicrosphere's functionality and also to decode the dye ratios containedin those microspheres. The adhesion or affixing technique, however, mustnot chemically insulate the microspheres from the analyte.

FIGS. 5A and 5B are micrographs of the distal end 212 of the bundle 202illustrating the preferred technique for attaching the microspheres 10to the bundle 202. Wells 250 are formed at the center of each opticalfiber 252 of the bundle 202. As shown in FIG. 5B, the size of the wells250 are coordinated with the size of the microspheres 10 so that themicrospheres 10 can be placed within the wells 250. Thus, each opticalfiber 252 of the bundle 202 conveys light from the single microsphere 10contained in 5 its well. Consequently, by imaging the end of the bundle202 onto the CCD array 236, the optical signatures of the microspheres10 are individually interrogatable.

FIG. 6 illustrates how the microwells 250 are formed and microspheres 10placed in the wells. A 1 mm hexagonally-packed imaging fiber containsapproximately 20,600 individual optical fibers that have coresapproximately 3.7 μm across (Part No. ET26 from Galileo Fibers).Typically, the cores of each fiber are hexagonally shaped as a resultthe starting preform; that is, during drawing the fiber does not usuallychange shape. In some cases, the shape can be circular, however.

In step 270, both the proximal and distal ends 212,214 of the fiberbundle 202 are successively polished on 12 μm, 9 μm, 3 μm, 1 μm, and 0.3μm lapping films. Subsequently, the ends can be inspected for scratcheson an atomic force microscope. In step 272, a representative etching isperformed on the distal end 212 of the bundle 202. A solution of 0.2grams NH₄F (ammonium fluoride) with 600 μl distilled H₂O and 100 μl ofHF (hydrofluoric acid), 50% stock solution, may be used. The distal end212 is etched in this solution for a specified time, preferablyapproximately 30 to 600 seconds, with about 80 seconds being preferred.

Upon removal from this solution, the bundle end is immediately placed indeionized water to stop any further etching in step 274. The fiber isthen rinsed in running tap water. At this stage, sonication ispreferably performed for several minutes to remove any salt productsfrom the reaction. The fiber is then allowed to air dry.

The foregoing procedure produces wells by the anisotropic etching of thefiber cores 254 favorably with respect to the cladding 256 for eachfiber of the bundle 202. The wells have approximately the diameter ofthe cores 254, 3.7 μm. This diameter is selected to be slightly largerthan the diameters of the microspheres used, 3.1 μm, in the example. Thepreferential etching occurs because the pure silica of the cores 254etches faster in the presence of hydrofluoric acid than thegermanium-doped silica claddings 256.

The microspheres are then placed in the wells 250 in step 276 accordingto a number of different techniques. The placement of the microspheresmay be accomplished by dripping a solution containing the desiredrandomly mixed subpopulations of the microspheres over the distal end212, sonicating the bundle to settle the microspheres in the wells, andallowing the microsphere solvent to evaporate. Alternatively, thesubpopulations could be added serially to the bundle end. Microspheres10 may then be fixed into the wells 250 by using a dilute solution ofsulfonated Nafion that is dripped over the end. Upon solventevaporation, a thin film of Nafion was formed over the microsphereswhich holds them in place. This approach is compatible for fixingmicrospheres for pH indication that carry FITC functionality. Theresulting array of fixed microspheres retains its pH sensitivity due tothe permeability of the sulfonated Nafion to hydrogen ions. Thisapproach, however, can not be employed generically as Nafion isimpermeable to most water soluble species. A similar approach can beemployed with different polymers. For example, solutions of polyethyleneglycol, polyacrylamide, or polyhydroxymethyl methacrylate (polyHEMA) canbe used in place of Nafion, providing the requisite permeability toaqueous species.

An alternative fixation approach employs microsphere swelling to entrapeach microsphere 10 in its corresponding microwell 250. In thisapproach, the microspheres are first distributed into the microwells 250by sonicating the microspheres suspended in a non-swelling solvent inthe presence of the microwell array on the distal end 212. Afterplacement into the microwells, the microspheres are subsequently exposedto an aqueous buffer in which they swell, thereby physically entrappingthem, analogous to muffins rising in a muffin tin.

One of the most common microsphere formations is tentagel, astyrene-polyethylene glycol co-polymer. These microspheres are unswollenin nonpolar solvents such as hexane and swell approximately 20-40% involume upon exposure to a more polar or aqueous media. This approach isextremely desirable since it does not significantly compromise thediffusional or permeability properties of the microspheres themselves.

FIGS. 7A and 7B show polymer coated microspheres 12 in wells 250 aftertheir initial placement and then after tapping and exposure to airpulses. FIGS. 7A and 7B illustrate that there is no appreciable loss ofmicrospheres from the wells due to mechanical agitation even without aspecific fixing technique. This effect is probably due to electrostaticforces between the microspheres and the optical fibers. These forcestend to bind the microspheres within the wells. Thus, in mostenvironments, it may be unnecessary to use any chemical or mechanicalfixation for the microspheres.

In a preferred embodiment, particularly when wells are used, asonication step may be used to place beads in the wells.

It should be noted that not all sites of an array may comprise a bead;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onebead, although this is not preferred.

In some embodiments, for example when chemical attachment is done, it ispossible to attach the beads in a non-random or ordered way. Forexample, using photoactivatible attachment linkers or photoactivatibleadhesives or masks, selected sites on the array may be sequentiallyrendered suitable for attachment, such that defined populations of beadsare laid down.

In addition, since the size of the array will be set by the number ofunique optical signatures, it is possible to “reuse” a set of uniqueoptical signatures to allow for a greater number of test sites. This maybe done in several ways; for example, by using a positional codingscheme within an array; different sub-bundles may reuse the set ofoptical signatures. Similarly, one embodiment utilizes bead size as acoding modality, thus allowing the reuse of the set of unique opticalsignatures for each bead size. Alternatively, sequential partial loadingof arrays with beads can also allow the reuse of optical signatures.

In a preferred embodiment, a spatial or positional coding system isdone. In this embodiment, there are sub-bundles or subarrays (i.e.portions of the total array) that are utilized. By analogy with thetelephone system, each subarray is an “area code”, that can have thesame tags (i.e. telephone numbers) of other subarrays, that areseparated by virtue of the location of the subarray. Thus, for example,the same unique tags can be reused from bundle to bundle. Thus, the useof 50 unique tags in combination with 100 different subarrays can forman array of 5000 different bioactive agents. In this embodiment, itbecomes important to be able to identify one bundle from another; ingeneral, this is done either manually or through the use of markerbeads, i.e. beads containing unique tags for each subarray.

In alternative embodiments, additional encoding parameters can be added,such as microsphere size. For example, the use of different size beadsmay also allow the reuse of sets of optical signatures; that is, it ispossible to use microspheres of different sizes to expand the encodingdimensions of the microspheres. Optical fiber arrays can be fabricatedcontaining pixels with different fiber diameters or cross-sections;alternatively, two or more fiber optic bundles or arrays, each withdifferent cross-sections of the individual fibers, can be added togetherto form a larger bundle or array; or, fiber optic bundles or arrays withfiber of the same size cross-sections can be used, but just withdifferent sized beads. With different diameters, the largest wells canbe filled with the largest microspheres and then moving ontoprogressively smaller microspheres in the smaller wells until all sizewells are then filled. In this manner, the same dye ratio could be usedto encode microspheres of different sizes thereby expanding the numberof different oligonucleotide sequences or chemical functionalitiespresent in the array. Although outlined for fiber optic substrates, thisas well as the other methods outlined herein can be used with othersubstrates and with other attachment modalities as well.

In a preferred embodiment, the coding and decoding is accomplished bysequential loading of the microspheres into the array. As outlined abovefor spatial coding, in this embodiment, the optical signatures can be“reused”. In this embodiment, the library of microspheres eachcomprising a different bioactive agent (or the subpopulations eachcomprise a different bioactive agent), is divided into a plurality ofsublibraries; for example, depending on the size of the desired arrayand the number of unique tags, 10 sublibraries each comprising roughly10% of the total library may be made, with each sublibrary comprisingroughly the same unique tags. Then, the first sublibrary is added to thefiber optic bundle or array comprising the wells, and the location ofeach bioactive agent is determined, using its optical signature. Thesecond sublibrary is then added, and the location of each opticalsignature is again determined. The signal in this case will comprise the“first” optical signature and the “second” optical signature; bycomparing the two matrices the location of each bead in each sublibrarycan be determined. Similarly, adding the third, fourth, etc.sublibraries sequentially will allow the array to be filled.

Thus, arrays are made of a large spectrum of chemical functionalitiesutilizing the compositions of invention comprising microspheres andsubstrates with discrete sites on a surface. Specifically, prior artsensors which can be adapted for use in the present invention includefour broad classifications of microsphere sensors: 1) basic indicatorchemistry sensors; 2) enzyme-based sensors; 3) immuno-based sensors(both of which are part of a broader general class of protein sensors);and 4) geno-sensors.

In a preferred embodiment, the bioactive agents are used to detectchemical compounds. A large number of basic indicator sensors have beenpreviously demonstrated. Examples include:

TABLE III TARGET ANALYTE BIOACTIVE AGENT NOTES (λ_(AB)/λ_(EM)) pHSensors seminaphthofluoresceins e.g., carboxyl-SNAFL based on:seminaphthorhodafluors e.g., carboxyl-SNARF 8-hydroxypyrene-1,3,6-trisulfonic acid fluorescein CO2 Sensors seminaphthofluoresceins e.g.,carboxyl-SNAFL based On: seminaphthorhodafluors e.g., carbody-SNARF8-hydroxypyrene-1,3,6- trisulfonic acid Metal Ions desferriozamine Be.g., Fe Sensors cyclen derivative e.g., Cu, Zn based on: derivatizedpeptides e.g., FITC-Gly-Gly-His, and FITC-Gly His, Cu, Zn fluorexon(calcine) e.g., Ca, Mg, Cu, Pb, Ba calcine blue e.g., Ca, Mg, Cu methylcalcine blue e.g., Ca, Mg, Cu ortho-dianisidine e.g., Zn tetracetic acid(ODTA) bis-salicylidene e.g., Al ethylenediamine (SED)N-(6-methozy-8-quinolyl-p- e.g., Zn toluenesulfonamine (TSQ) Indo-1e.g., Mn, Ni Fura-2 e.g., Mn, Ni Magesium Green e.g., Mg, Cd, Tb O₂Siphenylisobenzofuran 409/476 Methoxyvinyl pyrene 352/401 Nitritediaminonaphthalene 340/377 NO luminol 355/411 dihydrohodamine 289/noneCa²⁺ Bis-fura 340/380 Calcium Green visible light/530 Fura-2 340/380Indo-1 405/485 Fluo-3 visible light/525 Rhod-2 visible light/570 Mg²⁺Mag-Fura-2 340/380 Mag-Fura-5 340/380 Mag-Indo-1 405/485 Magnesium Green475/530 Magnesium Orange visible light/545 Zn²⁺ Newport Green 506/535TSQ Methoxy-Quinobyl 334/385 Cu⁺ Phen Green 492/517 Na⁺ SBFI 339/565SBFO 354/575 Sodium Green 506/535 K⁺ PBFI 336/557 Cl⁻ SPQ 344/443 MQAE350/460Each of the chemicals listed in Table III directly produces an opticallyinterrogatable signal or a change in the optical signature, as is morefully outlined below, in the presence of the targeted analyte.

Enzyme-based microsphere sensors have also been demonstrated and couldbe manifest on microspheres. Examples include:

TABLE IV SENSOR TARGET Bioactive agent Glucose Sensor glucose oxidase(enz.) + O₂-sensitive dye (see Table I) Penicillin Sensor penicillinase(enz.) + pH-sensitive dye (see Table I) Urea Sensor urease (enz.) +pH-sensitive dye (see Table I) Acetylcholine Sensor acetylcholinesterase(enz.) + pH-sensitive dye (see Table I)Generally, as more fully outlined above, the induced change in theoptical signal due to the presence of the enzyme-sensitive chemicalanalyte occurs indirectly in this class of chemical functionalities. Themicrosphere-bound enzyme, e.g., glucose oxidase, decomposes the targetanalyte, e.g., glucose, consume a co-substrate, e.g., oxygen, or producesome by-product, e.g., hydrogen peroxide. An oxygen sensitive dye isthen used to trigger the signal change.

Immuno-based microsphere sensors have been demonstrated for thedetection for environmental pollutants such as pesticides, herbicides,PCB's and PAH's. Additionally, these sensors have also been used fordiagnostics, such as bacterial (e.g., leprosy, cholera, lyme disease,and tuberculosis), viral (e.g., HIV, herpes simplex, cytomegalovirus),fungal (e.g., aspergillosis, candidiasis, cryptococcoses), Mycoplasmal(e.g., mycoplasmal pneumonia), Protozoal (e.g., amoebiasis,toxoplasmosis), Rickettsial (e.g., Rocky Mountain spotted fever), andpregnancy tests.

Microsphere genosensors may also be made (see the Examples). These aretypically constructed by attaching a probe sequence to the microspheresurface chemistry, typically via an NH₂ group. A fluorescent dyemolecule, e.g., fluorescein, is attached to the target sequence, whichis in solution. The optically interrogatable signal change occurs withthe binding of the target sequences to the microsphere. This produces ahigher concentration of dye surrounding the microsphere than in thesolution generally. A few demonstrated probe and target sequences, seeFerguson, J. A. et al. Nature Biotechnology, Vol. 14, December 1996, arelisted below in Table V.

TABLE V PROBE SEQUENCES TARGET SEQUENCES B-glo(+) (segment of humanB-globin) 5′- B-glo(+)-CF NH₂-(CH₂)₈-)TT TTT TTT TCA ACT TCA5′-Fluorescein-TC AAC GTG GAT GAA TCC ACG TTC ACC-3 GTT C-3′IFNG(interferon gamma 1)5′-NH₂-(CH₂)₈- IFNG-CF T₁₂-TGG CTT CTC TTG GCTGTT ACT-3′ 5′-Fluorescein-AG TAA CAG CCA AGA GAA CCC AAA-3′IL2(interleukin-2)5′-NH₂-(CH₂)₈-T₁₂-TA IL2-CF ACC GAA TCC CAA ACT CACCAG-3′ 5′-Fluorescein-CT GGT GAG TTT GGG ATT CTT GTA-3′IL4(interleukin-4)5′NH₂-(CH₂)₈-T₁₂-CC IL4-CF AAC TGC TTC CCC CTC TGT-3′5′-Fluorescein-AC AGA GGG GGA AGC AGT TGG-3′IL6(interleukin-6)5′NH₂-(CH₂)₈-T12-GT IL6-CF TGG GTC AGG GGT GGT TATT-3′ 5′-Fluorescein-AA TAA CCA CCC CTG ACC CAA C-3′It should be further noted that the genosensors can be based on the useof hybridization indicators as the labels. Hybridization indicatorspreferentially associate with double stranded nucleic acid, usuallyreversibly. Hybridization indicators include intercalators and minorand/or major groove binding moieties. In a preferred embodiment,intercalators may be used; since intercalation generally only occurs inthe presence of double stranded nucleic acid, only in the presence oftarget hybridization will the label light up.

The present invention may be used with any or all of these types ofsensors. As will be appreciated by those in the art, the type andcomposition of the sensor will vary widely, depending on the compositionof the target analyte. That is, sensors may be made to detect nucleicacids, proteins (including enzyme sensors and immunosensors), lipids,carbohydrates, etc; similarly, these sensors may include bioactiveagents that are nucleic acids, proteins, lipids, carbohydrates, etc. Inaddition, a single array sensor may contain different binding ligandsfor multiple types of analytes; for example, an array sensor for HIV maycontain multiple nucleic acid probes for direct detection of the viralgenome, protein binding ligands for direct detection of the viralparticle, immuno-components for the detection of anti-HIV antibodies,etc.

In addition to the beads and the substrate, the compositions of theinvention may include other components, such as light sources, opticalcomponents such as lenses and filters, detectors, computer componentsfor data analysis, etc.

The arrays of the present invention are constructed such thatinformation about the identity of the bioactive agent is built into thearray, such that the random deposition of the beads on the surface ofthe substrate can be “decoded” to allow identification of the bioactiveagent at all positions. This may be done in a variety of ways.

In a preferred embodiment, the beads are loaded onto the substrate andthen the array is decoded, prior to running the assay. This is done bydetecting the optical signature associated with the bead at each site onthe array. This may be done all at once, if unique optical signaturesare used, or sequentially, as is generally outlined above for the“reuse” of sets of optical signatures. Alternatively, decoding may occurafter the assay is run.

Once made and decoded if necessary, the compositions find use in anumber of applications. Generally, a sample containing a target analyte(whether for detection of the target analyte or screening for bindingpartners of the target analyte) is added to the array, under conditionssuitable for binding of the target analyte to at least one of thebioactive agents, i.e. generally physiological conditions. The presenceor absence of the target analyte is then detected. As will beappreciated by those in the art, this may be done in a variety of ways,generally through the use of a change in an optical signal. This changecan occur via many different mechanisms. A few examples include thebinding of a dye-tagged analyte to the bead, the production of a dyespecies on or near the beads, the destruction of an existing dyespecies, a change in the optical signature upon analyte interaction withdye on bead, or any other optical interrogatable event.

In a preferred embodiment, the change in optical signal occurs as aresult of the binding of a target analyte that is labeled, eitherdirectly or indirectly, with a detectable label, preferably an opticallabel such as a fluorochrome. Thus, for example, when a proteinaceoustarget analyte is used, it may be either directly labeled with a fluor,or indirectly, for example through the use of a labeled antibody.Similarly, nucleic acids are easily labeled with fluorochromes, forexample during PCR amplification as is known in the art. Alternatively,upon binding of the target sequences, an intercalating dye (e.g.ethidium bromide) can be added subsequently to signal the presence ofthe bound target to the probe sequence. Upon binding of the targetanalyte to a bioactive agent, there is a new optical signal generated atthat site, which then may be detected. Alternatively, in some cases, asdiscussed above, the target analyte such as an enzyme generates aspecies (for example, a fluorescent product) that is either directly orindirectly detectable optically.

Furthermore, in some embodiments, a change in the optical signature maybe the basis of the optical signal. For example, the interaction of somechemical target analytes with some fluorescent dyes on the beads mayalter the optical signature, thus generating a different optical signal.For example, fluorophore derivatized receptors may be used in which thebinding of the ligand alters the signal.

As will be appreciated by those in the art, in some embodiments, thepresence or absence of the target analyte may be done using changes inother optical or non-optical signals, including, but not limited to,surface enhanced Raman spectroscopy, surface plasmon resonance,radioactivity, etc.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins, e.g. albumin, detergents, etc which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

In a preferred embodiment, the compositions are used to probe a samplesolution for the presence or absence of a target analyte. By “targetanalyte” or “analyte” or grammatical equivalents herein is meant anyatom, molecule, ion, molecular ion, compound or particle to be eitherdetected or evaluated for binding partners. As will be appreciated bythose in the art, a large number of analytes may be used in the presentinvention; basically, any target analyte can be used which binds abioactive agent or for which a binding partner (i.e. drug candidate) issought.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. When detection of a target analyte is done, suitabletarget analytes include, but are not limited to, an environmentalpollutant (including pesticides, insecticides, toxins, etc.); a chemical(including solvents, polymers, organic materials, etc.); therapeuticmolecules (including therapeutic and abused drugs, antibiotics, etc.);biomolecules (including hormones, cytokines, proteins, nucleic acids,lipids, carbohydrates, cellular membrane antigens and receptors (neural,hormonal, nutrient, and cell surface receptors) or their ligands, etc);whole cells (including procaryotic (such as pathogenic bacteria) andeukaryotic cells, including mammalian tumor cells); viruses (includingretroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); andspores; etc. Particularly preferred analytes are nucleic acids andproteins.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected or evaluated forbinding partners using the present invention. Suitable protein targetanalytes include, but are not limited to, (1) immunoglobulins; (2)enzymes (and other proteins); (3) hormones and cytokines (many of whichserve as ligands for cellular receptors); and (4) other proteins.

In a preferred embodiment, the target analyte is a nucleic acid. Theseassays find use in a wide variety of applications.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

The present invention also finds use as a methodology for the detectionof mutations or mismatches in target nucleic acid sequences. Forexample, recent focus has been on the analysis of the relationshipbetween genetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants. There area number of well known polymorphisms that cause clinically importantphenotypes; for example, the apoE2/3/4 variants are associated withdifferent relative risk of Alzheimer's and other diseases (see Cordor etal., Science 261(1993). Multiplex PCR amplification of SNP loci withsubsequent hybridization to oligonucleotide arrays has been shown to bean accurate and reliable method of simultaneously genotyping at leasthundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see alsoSchafer et al., Nature Biotechnology 16:33-39 (1998). The compositionsof the present invention may easily be substituted for the arrays of theprior art.

In a preferred embodiment, the compositions of the invention are used toscreen bioactive agents to find an agent that will bind, and preferablymodify the function of, a target molecule. As above, a wide variety ofdifferent assay formats may be run, as will be appreciated by those inthe art. Generally, the target analyte for which a binding partner isdesired is labeled; binding of the target analyte by the bioactive agentresults in the recruitment of the label to the bead, with subsequentdetection.

In a preferred embodiment, the binding of the bioactive agent and thetarget analyte is specific; that is, the bioactive agent specificallybinds to the target analyte. By “specifically bind” herein is meant thatthe agent binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding, although insome embodiments, wash steps are not desired; i.e. for detecting lowaffinity binding partners. In some embodiments, for example in thedetection of certain biomolecules, the dissociation constants of theanalyte to the binding ligand will be less than about 10⁴-10⁻⁶ M⁻¹, withless than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and less than about10⁻⁷-10⁻⁹ M⁻¹ being particularly preferred.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentireity.

EXAMPLES Example 1: Enzyme-Based Sensor

Subpopulation A

-   -   Bioactive agent: Alkaline phosphatase    -   Target substrate: fluorescein diphosphate (FDP)    -   Reported dye ratio: 1:1 ratio of DiIC:TRC, where DiIC is        1,1′,3,3,3′,3′-hexamethyl-indodicarbocyanine iodide and TRC is        Texas Red cadaverine        A range of ratios of light intensities are selected that are        representative of the optical signature for the dye ratio of the        subpopulation based on the quantum yield of the two dyes. The        optical signature for this subpopulation is:        $\frac{{iIC}\quad\lambda\quad\text{intensity-ave.}\quad{DiIC}\quad{background}}{{TRC}\quad\lambda\quad{\text{intensity-ave} \cdot {TRC}}\quad{background}} = {0.847 \pm 0.23}$        Subpopulation B    -   Bioactive agent: B-Galactosidase;    -   Target substrate=fluorescein di-B-galactopyranoside (FDG)    -   Reporter dye ratio: 10:1 ratio of DiIC:TRC which translates to        an optical signature of:        $\frac{{DiIC}\quad\lambda\quad{\text{intensity-ave}.\quad{DiIC}}\quad{background}}{{TRC}\quad\lambda\quad{\text{intensity-ave}.\quad{TRC}}\quad{background}} = {4.456 \pm 1.27}$        Subpopulation C    -   Bioactive agent: B-glucuronidase    -   Target substrate=fluorescein di-B-D-glucuronide (FDGicu).    -   Reporter dye ratio: 1:10 ratio of DiIC:TRC, which translates to        an optical signature of:        $\frac{{DiIC}\quad A\quad{\text{intensity-ave}.\quad{DiIC}}\quad{background}}{{TRC}\quad A\quad{\text{intensity-ave}.\quad{TRC}}\quad{background}} = {0.2136 + 0.03}$

When the microsphere populations are in the presence of one or more ofthe substrates, the respective enzymes on the microspheres catalyze thebreakdown of the substrates producing fluorescein which is fluorescent,emitting light at 530 nanometers when excited at 490 nm. The productionof fluorescein localized to particular beads is then monitored. In thisapproach, the localization of fluorescein around the microspheres isincreased by using a substrate solution of 90% glycerol and 10%substrate. The glycerol inhibits the generated fluorescein fromdiffusing away from the microsphere reaction sites.

During the experiment, images in the encoded wavelengths are firsttaken. Since both DiIC and TRC are excited at 577 nm. Each microsphere'semissions at 670 nm, indicative of the presence of DiIC and 610 nmindicative of the presence of TRC were recorded using a 595 nm dichroicand an acquisition time of 5 seconds for the CCD 236. Next, the distalend 212 of the fiber bundle is placed in a buffer and another imagetaken while illuminating the beams with 490 nm light. Emissions in the530 nm fluorescein wavelengths were recorded with a 505 nm dichroic. Inthis case, a CCD acquisition time of one second was used. This processprovides a background normalizing image. The buffer was removed and thefiber allowed to dry to avoid substrate solution dilution.

The substrate solution is then introduced and CCD images acquired every30 seconds to a minute for 30 minutes While illuminating themicrospheres with 490 nm light and collecting emissions in the 530 nmrange. Fiber is then placed back in the buffer solution and anotherbackground image captured. Those beads that generate a signal indicativeof fluorescein production are decoded. Depending in the ratio of theintensity of light from the two reporter dyes, DiIC:TRC, the bioactiveagent of the optically active beads may be decoded according to thefollowing table.

0.617-1.08 alkaline phosphatase bead 3.188-5.725 β-galactosidase bead0.183-0.243 β-glucunonidese beadThis process is then repeated for the remaining two substrates.

FIGS. 8A-8C are images generated by the CCD 236 when the beadpopulations are exposed to fluorescein diphosphate. FIG. 8A illustratesthe signals from the alkaline phosphatase microspheres when excited at490 nm and recording emissions at 530 nm, emissions at this wavelengthbeing indicative of fluorescein production. FIG. 8B shows the imagecaptured by the CCD when the microspheres are excited at 577 nm andemissions at 670 nm are recorded. This wavelength is an encodingwavelength indicative of the concentration of DiIC on the microspheres.Finally, FIG. 8C shows the image when the microspheres are excited with577 nm light and emissions in the 610 nm range are recorded beingindicative of the concentration of TRC in the microspheres.

In a similar vein, FIGS. 9A and 9B are images when the microspheres areexposed to fluorescein β-d-galactosidase. FIG. 9A shows emissions at 530nm indicative of the fluorescein production; and FIG. 9B shows lightemitted at the 670 nm range indicative of the presence of DiIC.

These micrographs, FIG. 8A-8C and 9A-9B illustrate that fluoresceinproduction around the microspheres may be detected as an opticalsignature change indicative of reactions involving the bioactive agentof the microspheres. The micrographs also illustrate that the opticalsignatures may be decoded to determine the chemical functionalities oneach microsphere.

Immunosensor

Three separate subpopulations of beads were used. In subpopulation A,xrabbit antibodies (Ab) were affixed to the surface of the microspheres;in subpopulation B, xgoat antibodies were affixed to the microspheres;and in subpopulation C, xmouse antibodies were affixed to themicrospheres. These three separate subpopulations were identified usinga DiIC:TRC encoding ratio similar to that in the previously describedexperiment.

For the first step of the experiment, images at the encoded wavelengthswere captured using 577 nm excitation and looking for emissions at 610and 670 nm. After this decoding, the fiber was placed in a buffer and animage taken at 530 nm with 490 nm excitation. This provided a backgroundnormalizing signal at the fluorescein emission wavelength. Next, thefiber was placed in rabbit IgG antigen (Ag) which is fluoresceinlabeled. Images were then captured every few minutes at the 530 nmemission wavelength for fluorescein. FIGS. 10A and 10B are micrographsshowing the image captured by the CCD prior to and subsequent toexposure to a rabbit antigen, which clearly show reaction of theselected micropheres within the population.

Note, if the fluorescein background from the antigen solution is toohigh to see the antibody-antigen signal, the fiber bundle may be placedin a buffer. This removes the background florescence leaving only theAb-Ag signal.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A composition comprising: a) a substrate with a surface comprisingdiscrete sites at a density of at least 100 discrete sites per 1 mm²,said discrete sites comprising wells; and b) a population ofmicrospheres randomly distributed in said wells, said populationcomprising at least a first and a second subpopulation, saidmicrospheres comprising a bioactive agent, and wherein said sites canhave only a single microsphere.
 2. A composition comprising: a) asubstrate with a patterned surface comprising discrete sites, saidsubstrate comprising discrete sites at a density of at least 100discrete sites per 1 mm²; and b) a population of microspheres, randomlydistributed on said sites, wherein each microsphere comprises abioactive agent; and wherein said sites can have only a singlemicrosphere.
 3. A composition according to claim 1 or 2 wherein saidsubstrate is a fiber optic bundle.
 4. A composition according to claim 1or 2 wherein said substrate is selected from the group consisting ofglass and plastic.
 5. A composition according to claim 1 wherein saidpopulation of microspheres comprises at least a first and a secondsubpopulation, wherein the microspheres of said first subpopulation ofmicrospheres are a different size than the microspheres of said secondsubpopulation.
 6. A composition according to claim 1 or 2 wherein saidbioactive agent comprises a protein.
 7. A composition according to claim6 wherein said protein is selected from the group consisting of enzymesand antibodies.
 8. A composition according to claim 1 or 2 wherein saidbioactive agent is a nucleic acid.
 9. A composition according to claim2, wherein said population of microspheres comprises at least a firstand a second subpopulation.
 10. A composition according to claim 9,wherein the microspheres of said first subpopulation of microspheres area different size than the microspheres of said second subpopulation. 11.A composition according to claim 1, 9, 5, or 10 wherein said first andsaid second subpopulations comprise a first and a second bioactiveagent, respectively.
 12. The composition according to claim 11, whereinsaid first and second subpopulations further comprise a first and asecond optical signature, respectively.
 13. A composition according toclaim 12 wherein said at least one of said optical signatures comprisesat least one chromophore.
 14. A composition according to claim 12wherein said at least one of said optical signatures comprises at leastone fluorescent dye.
 15. A composition according to claim 14 whereinsaid fluorescent dye is entrapped within said microspheres.
 16. Acomposition according to claim 14 wherein said fluorescent dye isattached to said microspheres.
 17. A composition according to claim 12wherein said optical signature comprises at least two fluorescent dyes.18. A composition according to claim 9 wherein said bioactive agentcomprises a protein.
 19. A composition according to claim 18 whereinsaid protein is selected from the group consisting of enzymes andantibodies.
 20. A composition according to claim 9 wherein saidbioactive agent is a nucleic acid.
 21. A composition according to claim1 or 2 wherein said bead is covalently associated with the well.
 22. Acomposition according to claim 1 or 2 wherein said bead isnon-covalently associated with the well.
 23. A composition according toclaim 4 wherein said substrate is glass.
 24. A composition according toclaim 4 wherein said substrate is plastic.
 25. A composition accordingto claim 7 wherein said protein is an enzyme.
 26. A compositionaccording to claim 7 wherein said protein is an antibody.
 27. Acomposition according to claim 19 wherein said protein is an enzyme. 28.A composition according to claim 19 wherein said protein is an antibody.29. A method of determining the presence of at least a first and secondtarget analyte in a sample comprising: a) contacting said sample with acomposition comprising: i) a substrate with a patterned surfacecomprising discrete sites; and ii) a population of microspherescomprising at least a first and a second subpopulation, wherein saidfirst subpopulation comprises a first bioactive agent and said secondsubpopulation comprises a second bioactive agent, wherein saidmicrospheres are randomly distributed on said surface such that saiddiscrete sites contain only one microsphere; and b) determining thepresence of said first and second target analyte.
 30. A method accordingto claim 29 wherein said substrate is a optical fiber bundle and saidmicrospheres are located within wells at a first terminal end of saidbundle.
 31. A method according to claim 29 further comprisingidentifying the location of said first and second bioactive agent onsaid substrate.
 32. The method according to claim 29, wherein saiddiscrete sites are wells.
 33. The method according to claim 29, whereinsaid substrate is selected from the group consisting of glass andplastic.
 34. A method of making a composition comprising: a) providing apatterned surface comprising individual sites on a substrate; b)randomly distributing microspheres on said surface such that saidindividual sites contain microspheres, wherein said sites can have onlya single microsphere, and wherein said microspheres comprise at least afirst and a second subpopulation comprising: i) a first and secondbioactive agent, respectively; and ii) a first and second opticalsignature, respectively; c) detecting said first and second opticalsignatures while said microspheres are distributed on said surface; andd) correlating the location of at least one individual site on the arraywith the bioactive agent at that particular site.
 35. A method accordingto claim 34, wherein said distributing comprises serially adding saidsubpopulations to said sites.
 36. A method according to claim 34,wherein said substrate is a fiber optic bundle.
 37. A method accordingto claim 34, wherein said substrate is selected from the groupconsisting of glass and plastic.
 38. A method according to claim 34,wherein said sites are wells.
 39. A method according to claim 29 or 34,wherein said bead is covalently attached to the well.
 40. A methodaccording to claim 29 or 34, wherein said bead is non-covalentlyattached to the well.
 41. A method according to claim 29 or 34, whereinsaid bioactive agent is a nucleic acid.
 42. A method according to claim33 or 37 wherein said substrate is glass.
 43. A method according toclaim 33 or 37 wherein said substrate is plastic.
 44. A method accordingto claim 29 or 34 when said bioactive agent is a protein.